scholarly journals The inhibition of mitochondrial dicarboxylate transport by inorganic phosphate, some phosphate esters and some phosphonate compounds

1974 ◽  
Vol 138 (2) ◽  
pp. 171-175 ◽  
Author(s):  
R. N. Johnson ◽  
J. B. Chappell
Keyword(s):  
Succinate Dehydrogenase ◽  
Inorganic Phosphate ◽  
Potent Inhibitor ◽  
Competitive Inhibitor ◽  
Phosphate Esters ◽  
Alternative Substrate ◽  
Succinate Oxidation ◽  
Phenyl Phosphate ◽  
Dicarboxylate Transport ◽  
Phosphate Carrier

1. Pi competitively inhibited succinate oxidation by intact uncoupled mitochondria in the presence of sufficient N-ethylmaleimide to block the phosphate carrier, with a Ki of 2.5mm. 2. Of a large number of phosphate esters and phosphonate compounds, phenyl phosphate and phenylphosphonate were found to inhibit competitively uncoupled succinate oxidation by intact but not broken mitochondria. By comparison, benzoate was a relatively weak competitive inhibitor of succinate oxidation by intact mitochondria but a relatively potent inhibitor of succinate dehydrogenase. 3. Phenyl phosphate and phenylphosphonate were non-penetrant, and inhibited Pi-dependent swelling of mitochondria suspended in isosmolar ammonium malate in a manner non-competitive with Pi. The inhibitors did not affect mitochondrial swelling when tested with Pi alone. 4. It is concluded that: (i) phenyl phosphate and phenylphosphonate behaved as non-penetrant analogues of Pi, since their inhibitory properties were in strict contrast with those of benzoate; (ii) phenyl phosphate and phenylphosphonate interacted with the dicarboxylate carrier but not with the phosphate carrier; (iii) Pi was effective as a competitive inhibitor of succinate oxidation because of its being either an alternative substrate for the dicarboxylate carrier or competitive with succinate for the intramitochondrial cations as proposed by Harris & Manger (1968).

scholarly journals A colorimetric determination of inositol monophosphates as an assay for d-glucose 6-phosphate–1l-myoinositol 1-phosphate cyclase

1970 ◽  
Vol 119 (2) ◽  
pp. 183-186 ◽  
Author(s):  
J. E. G. Barnett ◽  
R. E. Brice ◽  
D. L. Corina
Keyword(s):  
Inorganic Phosphate ◽  
Periodic Acid ◽  
Competitive Inhibitor ◽  
Phosphate Esters ◽  
Colorimetric Determination ◽  
Radiochemical Method ◽  
Chemical Assay

A rapid and convenient chemical assay for the enzyme d-glucose 6-phosphate–1l-myoinositol 1-phosphate cyclase is described. The 1l-myoinositol 1-phosphate formed enzymically was oxidized with periodic acid liberating inorganic phosphate, which was assayed. myoInositol 2-phosphate can be assayed in the same way. Glucose 6-phosphate and other primary phosphate esters gave only very small quantities of inorganic phosphate under the conditions described. The Km of the enzyme for d-glucose 6-phosphate, 7.5±2.5×10−4m, was identical with that measured by the radiochemical method. 2-Deoxy-d-glucose 6-phosphate was a powerful competitive inhibitor, Ki 2.0±0.5×10−5m, but was not a substrate for the enzyme.

scholarly journals The probable site of action of thenolytrifluoracetone on the respiratory chain

1977 ◽  
Vol 164 (3) ◽  
pp. 617-620 ◽  
Author(s):  
W J Ingledew ◽  
T Ohnishi
Keyword(s):  
Succinate Dehydrogenase ◽  
Respiratory Chain ◽  
Close Association ◽  
Succinate Oxidation ◽  
Paramagnetic Resonance ◽  
Spin Interactions ◽  
Site Of Action ◽  
Transfer Inhibitor ◽  
Probable Site ◽  
Electron Paramagnetic

1. It is shown that the electron-transfer inhibitor thenoyltrifluoroacetone abolishes a respiratory-chain electron-paramagnetic-resonance absorbance due to spin-spin interactions of ubisemiquinones at concentrations similar to those required for inhibition of succinate oxidation. 2. A specific site of interaction of thenoyltrifluoroacetone with the respiratory chain is proposed to be on the ubisemiquinone with which succinate dehydrogenase reacts. 3. Our results further demonstrate the close association of the HiPIP (high-potential iron-sulphur) centre of succinate dehydrogenase with ubisemiquinone.

Threonine metabolism in isolated rat hepatocytes

2001 ◽  
Vol 281 (6) ◽  
pp. E1300-E1307 ◽  
Author(s):  
James D. House ◽  
Beatrice N. Hall ◽  
John T. Brosnan
Keyword(s):  
Potent Inhibitor ◽  
Rat Hepatocytes ◽  
Competitive Inhibitor ◽  
Acid Uptake ◽  
Keto Acid ◽  
Glycine Cleavage System ◽  
Isolated Rat Hepatocytes ◽  
Threonine Dehydratase ◽  
Threonine Dehydrogenase ◽  
Glycine Cleavage

The removal of the 1-carbon of threonine can occur via threonine dehydrogenase or threonine aldolase, this carbon ending up in glycine to be liberated by the mitochondrial glycine cleavage system and producing CO2. Alternatively, in the threonine dehydratase pathway, the 1-carbon ends up in α-ketobutyrate, which is oxidized in the mitochondria to CO2. Rat hepatocytes, incubated in Krebs-Henseleit medium, were incubated with 0.5 mMl-[1-14C]threonine, and14CO2 production was measured. Added glycine (0.3 mM) marginally suppressed threonine oxidation. Cysteamine (0.5 mM), a potent inhibitor of the glycine cleavage system, reduced threonine oxidation to 65% of controls. However, α-cyanocinnamate (0.5 mM), a competitive inhibitor of mitochondrial α-keto acid uptake, reduced threonine oxidation to 35% of controls. These data provided strong evidence that ∼65% of threonine oxidation occurs through the glycine-independent threonine dehydratase pathway. Glucagon (10−7 M) increased threonine oxidation and stimulated threonine uptake by these cells. In summary, the majority of threonine oxidation occurs through the threonine dehydratase pathway in rat hepatocytes, and threonine oxidation is increased by glucagon, which also increases threonine's transport.

scholarly journals Inorganic Phosphate as an Important Regulator of Phosphatases

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Claudia Fernanda Dick ◽  
André Luiz Araújo Dos-Santos ◽  
José Roberto Meyer-Fernandes
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Metabolic Pathways ◽  
Inorganic Phosphate ◽  
Phosphate Esters ◽  
Regulation System ◽  
Pi Starvation ◽  
Important Regulator ◽  
Phosphatase System ◽  
The Difference

Cellular metabolism depends on the appropriate concentration of intracellular inorganic phosphate (Pi). Pi starvation-responsive genes appear to be involved in multiple metabolic pathways, implying a complex Pi regulation system in microorganisms and plants. A group of enzymes is required for absorption and maintenance of adequate phosphate levels, which is released from phosphate esters and anhydrides. The phosphatase system is particularly suited for the study of regulatory mechanisms because phosphatase activity is easily measured using specific methods and the difference between the repressed and derepressed levels of phosphatase activity is easily detected. This paper analyzes the protein phosphatase system induced during phosphate starvation in different organisms.

scholarly journals New properties of Bacillus subtilis succinate dehydrogenase altered at the active site. The apparent active site thiol of succinate oxidoreductases is dispensable for succinate oxidation

1989 ◽  
Vol 260 (2) ◽  
pp. 491-497 ◽  
Author(s):  
L Hederstedt ◽  
L O Hedén
Keyword(s):  
Bacillus Subtilis ◽  
Succinate Dehydrogenase ◽  
Active Site ◽  
Amino Acid Position ◽  
Biochemical Properties ◽  
Cysteine Residue ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Succinate Oxidation ◽  
E Coli

Mammalian and Escherichia coli succinate dehydrogenase (SDH) and E. coli fumarate reductase apparently contain an essential cysteine residue at the active site, as shown by substrate-protectable inactivation with thiol-specific reagents. Bacillus subtilis SDH was found to be resistant to this type of reagent and contains an alanine residue at the amino acid position equivalent to the only invariant cysteine in the flavoprotein subunit of E. coli succinate oxidoreductases. Substitution of this alanine, at position 252 in the flavoprotein subunit of B. subtilis SDH, by cysteine resulted in an enzyme sensitive to thiol-specific reagents and protectable by substrate. Other biochemical properties of the redesigned SDH were similar to those of the wild-type enzyme. It is concluded that the invariant cysteine in the flavoprotein of E. coli succinate oxidoreductases corresponds to the active site thiol. However, this cysteine is most likely not essential for succinate oxidation and seemingly lacks an assignable specific function. An invariant arginine in juxtaposition to Ala-252 in the flavoprotein of B. subtilis SDH, and to the invariant cysteine in the E. coli homologous enzymes, is probably essential for substrate binding.

scholarly journals The oxidative activities of membrane vesicles from Bacillus caldolyticus. Energy-dependence of succinate oxidation

1978 ◽  
Vol 170 (2) ◽  
pp. 395-405 ◽  
Author(s):  
Anthony G. Dawson ◽  
J. B. Chappell
Keyword(s):  
Cytochrome C ◽  
Succinate Dehydrogenase ◽  
Cytochrome B ◽  
Atp Hydrolysis ◽  
Membrane Vesicles ◽  
Protonmotive Force ◽  
Anaerobic Conditions ◽  
Succinate Oxidation ◽  
Ample Supply ◽  
Energy Conserving

1. The properties of membrane vesicles from the extreme thermophile Bacillus caldolyticus were investigated. 2. Vesicles prepared by exposure of spheroplasts to ultrasound contained cytochromes a, b and c, and at 50°C they rapidly oxidized NADH and ascorbate in the presence of tetramethyl-p-phenylenediamine. Succinate and l-malate were oxidized more slowly, and dl-lactate, l-alanine and glycerol 1-phosphate were not oxidized. 3. In the absence of proton-conducting uncouplers the oxidation of NADH was accompanied by a net translocation of H+ into the vesicles. Hydrolysis of ATP by a dicyclohexylcarbodi-imide-sensitive adenosine triphosphatase was accompanied by a similarly directed net translocation of H+. 4. Uncouplers (carbonyl cyanide p-trifluoromethoxyphenylhydrazone or valinomycin plus NH4+) prevented net H+ translocation but stimulated ATP hydrolysis, NADH oxidation and ascorbate oxidation. The last result suggested an energy-conserving site in the respiratory chain between cytochrome c and oxygen. 5. Under anaerobic conditions the reduction of cytochrome b by ascorbate (with tetramethyl-p-phenylenediamine) was stimulated by ATP hydrolysis, indicating an energy-conserving site between cytochrome b and cytochrome c. However, no reduction of NAD+ supported by oxidation of succinate, malate or ascorbate occurred, neither did it with these substrates in the presence of ATP under anaerobic conditions, suggesting that there was no energy-conserving site between NADH and cytochrome b. 6. Succinate oxidation, in contrast with that of NADH and ascorbate, was strongly inhibited by uncouplers and stimulated by ATP hydrolysis. These effects were not observed when phenazine methosulphate, which transfers electrons from succinate dehydrogenase directly to oxygen, was present. It was concluded that in these vesicles the oxidation of succinate was energy-dependent and that the reoxidation of reduced succinate dehydrogenase was dependent on the outward movement of H+ by the protonmotive force. 7. In support of the foregoing conclusion it was shown that the reduction of fumarate by NADH was an energy-conserving process. 8. If the activities of vesicles accurately represent those of the intact organism it appears that in B. caldolyticus the reduction of fumarate to succinate at the expense of reducing equivalents from NADH is energetically favoured over succinate oxidation even under aerobic conditions. This may be related to the need for an ample supply of succinate for haem synthesis in order to provide cytochromes for the organism.

scholarly journals Succinate dehydrogenase functioning by a reverse redox loop mechanism and fumarate reductase in sulphate-reducing bacteria

Microbiology ◽  
2006 ◽  
Vol 152 (8) ◽  
pp. 2443-2453 ◽  
Author(s):  
Tanja Zaunmüller ◽  
David J. Kelly ◽  
Frank O. Glöckner ◽  
Gottfried Unden
Keyword(s):  
Succinate Dehydrogenase ◽  
Draft Genome ◽  
Desulfovibrio Desulfuricans ◽  
Geobacter Sulfurreducens ◽  
Fumarate Reductase ◽  
Desulfovibrio Vulgaris ◽  
Succinate Oxidation ◽  
Proton Potential ◽  
Fumarate Respiration ◽  
Reducing Bacteria

Sulphate- or sulphur-reducing bacteria with known or draft genome sequences (Desulfovibrio vulgaris, Desulfovibrio desulfuricans G20, Desulfobacterium autotrophicum [draft], Desulfotalea psychrophila and Geobacter sulfurreducens) all contain sdhCAB or frdCAB gene clusters encoding succinate : quinone oxidoreductases. frdD or sdhD genes are missing. The presence and function of succinate dehydrogenase versus fumarate reductase was studied. Desulfovibrio desulfuricans (strain Essex 6) grew by fumarate respiration or by fumarate disproportionation, and contained fumarate reductase activity. Desulfovibrio vulgaris lacked fumarate respiration and contained succinate dehydrogenase activity. Succinate oxidation by the menaquinone analogue 2,3-dimethyl-1,4-naphthoquinone depended on a proton potential, and the activity was lost after degradation of the proton potential. The membrane anchor SdhC contains four conserved His residues which are known as the ligands for two haem B residues. The properties are very similar to succinate dehydrogenase of the Gram-positive (menaquinone-containing) Bacillus subtilis, which uses a reverse redox loop mechanism in succinate : menaquinone reduction. It is concluded that succinate dehydrogenases from menaquinone-containing bacteria generally require a proton potential to drive the endergonic succinate oxidation. Sequence comparison shows that the SdhC subunit of this type lacks a Glu residue in transmembrane helix IV, which is part of the uncoupling E-pathway in most non-electrogenic FrdABC enzymes.

scholarly journals Reconstitution of the hexose phosphate translocator from the envelope membranes of wheat endosperm amyloplasts

1996 ◽  
Vol 319 (3) ◽  
pp. 717-723 ◽  
Author(s):  
Ian J TETLOW ◽  
Caroline G BOWSHER ◽  
Michael J EMES
Keyword(s):  
Inorganic Phosphate ◽  
Competitive Inhibitor ◽  
Dihydroxyacetone Phosphate ◽  
Carrier Protein ◽  
Sugar Phosphate ◽  
Wheat Endosperm ◽  
Single Carrier ◽  
Phosphate Translocator ◽  
Envelope Membranes ◽  
Hexose Phosphates

Amyloplasts were isolated and purified from wheat endosperm and the envelope membranes reconstituted into liposomes. Envelope membranes were solubilized in n-octyl β-D-glucopyranoside and mixed with liposomes supplemented with 5.6 mol% cholesterol to produce proteoliposomes of defined size, which showed negligible leakage of internal substrates. Transport experiments with proteoliposomes revealed a counter-exchange of glucose 1-phosphate (Glc1P), glucose 6-phosphate (Glc6P), inorganic phosphate (Pi), 3-phosphoglycerate and dihydroxyacetone phosphate. The Glc1P/Pi counter-exchange reaction exhibited an apparent Km for Glc1P of 0.4 mM. Glc6P was a competitive inhibitor of Glc1P transport (Ki 0.8 mM), and the two hexose phosphates could exchange with each other, indicating the operation of a single carrier protein. Glc1P/Pi antiport in proteoliposomes showed an exchange stoichiometry at pH 8.0 of 1 mol of phosphate transported per mol of sugar phosphate.

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